Deoxyribonuclease (DNase) is a phosphodiesterase capable of hydrolyzing polydeoxyribonucleic acid, and is known to occur in several molecular forms. Based on their biochemical properties and enzymatic activities, DNase proteins have been classified as two types, DNase I and DNase II. DNase I proteins have a pH optimum near neutrality, an obligatory requirement for divalent cations, and produce 5'-phosphate nucleotides on hydrolysis of DNA. DNase II proteins exhibit an acid pH optimum, do not require divalent cations for activity, and produce 3'-phosphate nucleotides on hydrolysis of DNA.
DNase from various species have been purified to a varying degree. For example, various forms of bovine DNase I have been purified and completely sequenced (Liao, et al., J. Biol. Chem. 248:1489-1495 (1973); Oefner, et al., J. Mol. Biol. 192:605-632 (1986); Lahm, et al., J. Mol. Biol. 221:645-667 (1991)), and DNA encoding bovine DNase I has been cloned and expressed (Worrall, et al., J. Biol. Chem 265:21889-21895 (1990)). Porcine and orcine DNase I proteins also have been purified and completely sequenced (Paudel, et al., J. Biol. Chem. 261:16006-16011 (1986); Paudel, et al., J. Biol. Chem. 261:16012-16017 (1986)).
DNA encoding a human DNase I has been isolated and sequenced and the DNA has been expressed in recombinant host cells, thereby enabling the production of human DNase I in commercially useful quantities. Shak, et al., Proc. Natl. Acad. Sci. 87:9188-9192 (1990). The term "human DNase I" will be used hereafter to refer to the mature polypeptide disclosed in Shak, et al.
DNA encoding other polypeptides having homology to human DNase I also have been identified. Rosen, et al., PCT Patent Publication No. WO 95/30428, published Nov. 16, 1995; Parrish, et al., Hum. Mol. Genet. 4:1557-1564 (1995); Baker, et. al., U.S. Patent Application No. 08/597,078 (filed Feb. 5, 1996).
DNase I has a number of known utilities and has been used for therapeutic purposes. Its principal therapeutic use has been to reduce the viscoelasticity of pulmonary secretions (including mucus) in such diseases as pneumonia and cystic fibrosis (CF), thereby aiding in the clearing of respiratory airways. See e.g., Lourenco, et al., Arch. Intern. Med. 142:2299-2308 (1982); Shak, et al., Proc. Natl. Acad. Sci. 87:9188-9192 (1990); Hubbard, et al., New Engl. J. Med. 326:812-815 (1992); Fuchs, et al., New Engl. J. Med. 331:637-642 (1994); Bryson, et al., Drugs 48:894-906 (1994). Mucus also contributes to the morbidity of chronic bronchitis, asthmatic bronchitis, bronchiectasis, emphysema, acute and chronic sinusitis, and even the common cold. DNase I is effective in reducing the viscoelasticity of pulmonary secretions by hydrolyzing, or degrading, high-molecular-weight DNA that is present in such secretions. Shak, et al., Proc. Natl. Acad. Sci. 87:9188-9192 (1990); Aitken, et al., J. Am. Med. Assoc. 267:1947-1951 (1992).
Various forms of DNase II also have reportedly been purified, including bovine DNase II (Lesca, J. Biol. Chem. 251:116-123 (1976)), human DNase II (Yamanaku, et al., J. Biol. Chem. 249:3884-3889 (1974); Murai, et al., J. Biochem. 87:1097-1103 (1980); Harosh, et al., Eur. J. Biochem. 202:479-484 (1991); Yasuda, et al., Biochem. Biophys. Acta 1119:185-193 (1992)), porcine DNase II (Bernardi, et al., Biochemistry 4:1725-1729 (1965); Liao, et al., J. Biol. Chem. 260:10708-10713 (1990)), and rat DNase II (Dulaney, et al., J. Biol. Chem. 247:1424-1432 (1972)). The physical properties of the human DNase II proteins described in these reports vary considerably (e.g., reported molecular weights range from 32,000 to 45,000 Daltons), which leads to uncertainty whether there is one or multiple naturally occurring forms of the human protein.
Recent interest in human DNase II has arisen because of its possible role in the programmed cell death process of apoptosis (Barry, et al., Arch. Biochem. Biophys. 300:440-450 (1993); Barry, et al., Cancer Res. 53:2349-2357 (1993)). One of the events that is characteristic of that process is the degradation of nuclear DNA into nucleosomal fragments. The ability to prevent or inhibit the expression of human DNase II or its enzymatic activity within human cells may be important in preventing or limiting such intracellular destruction of DNA, and thus may be an effective means of interrupting the process of apoptosis. In other instances, it may be useful to increase the expression of human DNase II within a certain population of cells within a human patient, such as cancer cells, in order to induce apoptosis of those cells.